Since nitrogen mustard derivatives continue to be at the forefront of cancer chemotherapy, we are continuing to investigate the molecular mechanisms of actions of these drugs, with the view that even a modest increase in selectivity of drugs designed on the basis of new mechanistic insights could have significant clinical impact. The current focus is on nitrogen mustard derivatives containing ring systems that bind to DNA by intercalation, since this is the most direct way in which these drugs could be targeted to specific DNA regions. Intercalating mustards are also of interest because they need only a monoalkylating group for cytotoxic and antitumor activities, indicating that they are effective without forming covalent crosslinks. A homologous series of intercalating mustards is being studied as a function of the length of the hydrocarbon chain connecting the intercalating (acridine) group with a mono or a bifunctional nitrogen mustard moiety. Substantial changes in base sequence selectivity have been found depending on the connecting chain length. Current studies attempt to relate both sequence selectivity and the production of different types of DNA lesions to cell killing potency. Although we have so far studied sequence selectivity only on purified DNA, we are developing methods to study this in cell nuclei. In addition, we are studying the formation of interstrand crosslinks in synthetic oligonucleotide duplexes, in order to determine the DNA sequence dependence and the effect of drug structure. We are also studying the production and repair of DNA lesions, such as interstrand and DNA-protein crosslinks and DNA strand breaks measured by DNA alkaline elution assays, in drug-treated cells. We find that the intercalating mustards are unusual, compared to the common nitrogen mustards, in that DNA strand break production is prominent and interstrand crosslinks are not detected. Intercalating mustards therefore may have a unique mechanism of action.